Methods for detection of anti-cytomegalovirus neutralizing antibodies

ABSTRACT

The present disclosure provides methods useful for determining levels of HCMV infection in host cells and, by extension, determining levels of neutralizing antibodies present in a sample. The present disclosure encompasses the recognition that HCMV viruses that have a fluorescent moiety permit detection of viral infection (e.g., by assessing fluorescence in cells after contacting the host cell with the virus). In some embodiments, levels of HCMV infection are determined by fluorescence detection where the virus has been preincubated with a test sample (e.g., a serum sample) from a subject. In some embodiments, the subject has been administered a candidate HCMV vaccine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 61/616,204, filed on Mar. 27, 2012, the contents of which are hereinincorporated by reference in their entirety.

BACKGROUND

Human cytomegalovirus (HCMV), a β-herpesvirus, is a ubiquitouslyoccurring pathogen. In an immunocompetent person, HCMV infection isnormally unnoticed, having at most mild and nonspecific symptoms. Bycontrast, certain risk groups, for example in immunosuppressed patientssuch as AIDS patients or transplant recipients, and after prenatalinfection, HCMV infection has serious manifestations (Staras S A et al.,2006 Clin Infect Dis 43(9):1143-51; Hebart H et al., 2004 Hum Immunol65(5):432-6; Rowshani A T et al., 2005 Transplantation 79(4):381-6).Existing therapies include the use of immunoglobulins and anti-viralagents such as ganciclovir and its derivatives, which are most effectivewhen used prophylactically or very early during infection in at riskpopulations. However, existing therapies are characterized bysignificant toxicity and limited efficacy, especially for late-onsetdisease (Boeckh M., 2004 Pediatr Transplant 8(Suppl. 5):19-27; Limaye AP., 2004 Transplantation 78(9): 1390-6), and they have not had an impacton congenital HCMV disease. Development of an effective vaccine toprotect against HCMV disease is recognized as an important public healthpriority (Arvin A M et al., 2004 Clin Infect Dis 39(2):233-9).

In vitro assays are important tools to evaluate candidate vaccines fortheir ability to interfere with HCMV infection. For example,neutralization assays have been developed to study immune responses ininfected individuals as well as to assess vaccine immunogen candidatesin both clinical and preclinical trials. In the case of HCMV, antigenbinding ELISAs can measure antibodies specific for HCMV antigens;however, only an assay in which neutralization of viral entry into cellsis measured can establish and quantify the biological activity of HCMVantigen-specific antibodies (Abai et al., 2007 J Immunol Methods332(1-2):82-93). Typically, in such neutralization assays for HCMV, thedegree to which neutralizing antibodies reduce HCMV infection of cellsin the assay is determined by quantification of nuclei of infected cellsbased on expression of one or more viral proteins in the cell. Suchanalyses can be time consuming and difficult to employ in highthroughput applications. There remains a need in the art for improvedmethods of screening potential HCMV vaccine candidates for neutralizingantibody induction.

SUMMARY

Among other things, the present disclosure provides methods useful fordetermining levels of HCMV infection in host cells and, by extension,determining levels of neutralizing antibodies present in a sample. Thepresent disclosure encompasses the recognition that HCMV viruses thathave a fluorescent moiety permit detection of viral infection (e.g., byassessing fluorescence in cells after contacting the host cell with thevirus). In some embodiments, levels of HCMV infection are determined byfluorescence detection where the virus has been preincubated with a testsample (e.g., a serum sample) from a subject. In some embodiments, thesubject has been administered a candidate HCMV vaccine.

Other features, objects, and advantages of the present disclosure areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present disclosure, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the disclosure will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only, not for limitation.

FIG. 1 depicts exemplary ELISA anti-gB antibody titers afterimmunization with bivalent gB virus-like particles (VLPs) (gB/pp65 andgB/pp65+gH-G/pp65). A potent and sustained immunity is induced bybivalent gB VLPs in rabbits after a single immunization.

FIG. 2 depicts exemplary FACS analysis of GFP expression in fibroblastcells indicative of neutralizing antibody response induced with gB/pp65CMV VLPs in rabbits. Rabbits (n=6/group) were immunized (IM) twice atweeks 0 and 8 and bled 2 weeks later. Sera were pooled and tested atindicated dilutions in comparison to Cytogam™ at similar dilutionsagainst GFP-expressing CMV virus (TB40) in HFF fibroblasts. 100,000cells were collected during flow cytometric analysis of infected (GFP⁺)cells.

FIG. 3 depicts exemplary FACS analysis of GFP expression in fibroblastcells indicative of neutralizing antibody response induced with bivalentgB+gH CMV VLPs in rabbits. Rabbits (n=6/group) were immunized (IM) twiceat weeks 0 and 8 and bled 2 weeks later. Sera were pooled and tested atindicated dilutions in comparison to Cytogam™ at similar dilutionsagainst GFP-expressing CMV virus (TB40) in HFF fibroblasts. 100,000cells were collected during flow cytometric analysis of infected (GFP⁺)cells.

FIG. 4 depicts exemplary percent neutralizations in HFF-1 cells.Depicted are neutralizations for 15RA09 group 7 (monovalentgB-G/monovalent gH-G adjuvanted with alum) pooled samples at P1Vd14,P1Vd28, P1Vd42, P1Vd55, and P2Vd14; and 15RA05 group 8 (empty MLV Gag)at P2Vd14, in presence of 10% guinea pig complement against 1:6CMV-GFP-TB40-010512 in HFF-1 cells.

FIG. 5 depicts exemplary percent neutralizations in ARPE-19 cells.Depicted are neutralizations for 15RA09 group 7 (monovalentgB-G/monovalent gH-G adjuvanted with alum) pooled samples at P1Vd14,P1Vd28, P1Vd42, P1Vd55, and P2Vd14; and 15RA05 group 8 (empty MLV Gag)at P2Vd14, in presence of 2.5% rabbit complement against 1:3CMV-GFP-Towne-150612 in ARPE-19 cells.

DEFINITIONS

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an l-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Antigen: As used herein, the term “antigen” refers to a substancecontaining one or more epitopes (either linear, conformational or both)that are recognized by antibodies. In certain embodiments, an antigen isor comprises a virus or a viral polypeptide. In some embodiments, theterm “antigen” refers to a subunit antigen (i.e., an antigen which isseparate and discrete from a whole virus with which the antigen isassociated in nature; e.g., an antigen which is associated with avirus-like particle). Alternatively or additionally, in someembodiments, the term “antigen” refers to killed, attenuated orinactivated viruses. In certain embodiments, an antigen is an“immunogen.”

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Amelioration: As used herein, the term “amelioration” is meant theprevention, reduction or palliation of a state, or improvement of thestate of a subject. Amelioration includes, but does not require completerecovery or complete prevention of a disease, disorder or condition(e.g., HCMV infection). The term “prevention” refers to a delay of onsetof a disease, disorder or condition. Prevention may be consideredcomplete when onset of a disease, disorder or condition has been delayedfor a predefined period of time.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic agent for thepatient to be treated. Each unit contains a predetermined quantity ofactive material calculated to produce the desired therapeutic effect. Itwill be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that arc administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regimen comprises a plurality ofdoses and at least two different time periods separating individualdoses.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end formation); (3) translation of an RNA into a polypeptide orprotein; and/or (4) post-translational modification of a polypeptide orprotein.

Fluorescence: As used herein, the term fluorescence refers to a moietythat luminesces. Typically fluorescent moieties contain electrons whichcan absorb electromagnetic energy at one wavelength and emitelectromagnetic energy at a second wavelength. Some proteins or smallmolecules in cells are naturally fluorescent (e.g., NADH, tryptophan,endogenous chlorophyll, phycoerythrin, or green fluorescent protein(GFP)). It will be appreciated that various mutants of fluorescentproteins have been engineered and may be used in accordance with thepresent disclosure, such as EGFP, blue fluorescent protein (EBFP, EBFP2,Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet),yellow fluorescent protein (YFP, Citrine, Venus, YPet), redox sensitiveGFP (roGFP), and monomeric GFP, among others. GFP and other fluorescentproteins can be expressed exogenously in cells alone or as a fusionprotein. This approach permits fluorescent proteins to be used asreporters for any number of biological events, such as subcellularlocalization and expression patterns.

Alternatively or additionally, specific or general proteins, nucleicacids, lipids or small molecules can be labeled with an extrinsicfluorophore, a fluorescent dye which can be a small molecule, protein orquantum dot. Exemplary fluorophores include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA);5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-I methylcoumarin;9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); AFPs—AutoFluorescent Protein—(QuantumBiotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™;Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™;Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™;Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin(APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; AminoactinomycinD; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC;APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B;Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ;Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue FluorescentProtein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst);bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy492/515;Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676. Bodipy Fl; Bodipy FLATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-Xconjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE;BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calccin;Calccin Blue; Calcium Crimson-; Calcium Green; Calcium Green-1 Ca.sup.2+Dye; Calcium Green-2 Ca.sup.2+; Calcium Green-5N Ca.sup.2+; CalciumGreen-C18 Ca.sup.2+; Calcium Orange; Calcofluor White;Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein);CFP/YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA;Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp;Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazinen; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM IMethylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8;Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl;Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE;Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3′DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123);Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP);Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer, DiD(Di1C18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (Di1C18(3)); IDinitrophenol; DiO (DiOC18(3)); DiR; DiR (Di1C18(7)); DM-NERF (high pH);DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP;ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidiumhomodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride;EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd InducedFluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC);Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold(Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™(high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl BrilliantRed B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow5GF; GeneBlazer, (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wildtype′ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP);GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258;Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine(FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1 lowcalcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); IntrawhiteCf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751(RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine;Lissamine Rhodamine B; Calcein/Ethidium homodimer, LOLO-1; LO-PRO-1;Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso TrackerGreen; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; MagnesiumGreen; Magnesium Orange; Malachite Green; Marina Blue; I MaxilonBrilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; MitotrackerRed; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i NuclearYellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488;Oregon Greecn™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline(Feulgen); PBFI; PE-CyS; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev;Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE];Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome BlueBlack; POPO-1; POPO-3; PO-PRO-1; PO-1 PRO-3; Primuline; Procion Yellow;Propidium lodid (P1); PyMPO; Pyrene; Pyronine; Pyronine B; PyrozalBrilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414;Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD;Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; RhodamineBB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine:Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine;R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI;Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron IBrilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glowBFP); sgGFP™ (super glow GFP); SITS (Primuline; StilbeneIsothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein;SNARFI; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange;Spectrum Red; SPQ (6-methoxy-N-(3 sulfopropyl)quinolinium); Stilbene;Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOXGreen; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); TexasRed™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine RedR; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3; YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductornanoparticles such as quantum dots; or caged fluorophore (which can beactivated with light or other electromagnetic energy source), or acombination thereof.

Fusion protein: As used herein, the term “fusion protein” generallyrefers to a polypeptide including at least two segments, each of whichshows a high degree of amino acid identity to a peptide moiety that (1)occurs in nature, and/or (2) represents a functional domain of apolypeptide. Typically, a polypeptide containing at least two suchsegments is considered to be a fusion protein if the two segments aremoieties that (1) are not included in nature in the same peptide, and/or(2) have not previously been linked to one another in a singlepolypeptide, and/or (3) have been linked to one another through actionof the hand of man.

Gene: As used herein, the term “gene” has its meaning as understood inthe art. It will be appreciated by those of ordinary skill in the artthat the term “gene” may include gene regulatory sequences (e.g.,promoters, enhancers, etc.) and/or intron sequences. It will further beappreciated that definitions of gene include references to nucleic acidsthat do not encode proteins but rather encode functional RNA moleculessuch as tRNAs, RNAi-inducing agents, etc. For the purpose of clarity wenote that, as used in the present application, the term “gene” generallyrefers to a portion of a nucleic acid that encodes a protein; the termmay optionally encompass regulatory sequences, as will be clear fromcontext to those of ordinary skill in the art. This definition is notintended to exclude application of the term “gene” to non-protein-codingexpression units but rather to clarify that, in most cases, the term asused in this document refers to a protein-coding nucleic acid.

Gene product or expression product: As used herein, the term “geneproduct” or “expression product” generally refers to an RNA transcribedfrom the gene (pre- and/or post-processing) or a polypeptide (pre-and/or post-modification) encoded by an RNA transcribed from the gene.

High-throughput: As used herein, the term “high-throughput” refersbroadly to investigations with a large number of assays such thatformatting of each individual sample, minimizing preparation steps andcomplications, and measuring of the assay results either in parallel orin rapid succession become important. High-throughput tests generally donot include manual, one-at-a-time assays, such as assays by a singleindividual in which the preparation, execution, measurement, and datacollection for one assay are all completed before the assay on the nextagent is done. High-throughput typically includes, for example, anyassays in which a batch of samples (e.g., 24, 96, 384 or more testsamples) are prepared and measured. Formatting the tests in such testsamples is meant to accelerate the assay process by enabling measurementin parallel or in rapid succession, such as with the assistance ofautomation.

Immunogenic: As used herein, the term “immunogenic” means capable ofproducing an immune response in a host animal against a non-host entity(e.g., an HCMV antigen). In certain embodiments, this immune responseforms the basis of the protective immunity elicited by a vaccine againsta specific infectious organism (e.g., an HCMV).

Immune response: As used herein, the term “immune response” refers to aresponse elicited in an animal. An immune response may refer to cellularimmunity, humoral immunity or may involve both. An immune response mayalso be limited to a part of the immune system. For example, in certainembodiments, an immunogenic composition may induce an increased IFNγresponse. In certain embodiments, an immunogenic composition may inducea mucosal IgA response (e.g., as measured in nasal and/or rectalwashes). In certain embodiments, an immunogenic composition may induce asystemic IgG response (e.g., as measured in serum). In certainembodiments, an immunogenic composition may induce virus-neutralizingantibodies or a neutralizing antibody response.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein.

Individual, subject, patient: As used herein, the terms “subject,”“individual” or “patient” refer to a human or a non-human mammaliansubject. The individual (also referred to as “patient” or “subject”)being treated is an individual (fetus, infant, child, adolescent, oradult) suffering from a disease, for example, HCMV infection. In someembodiments, the subject is at risk for HCMV infection. In someembodiments, the subject is an immunosuppressed subject. For example, insome embodiments, the immunosuppressed subject is selected from thegroup consisting of an HIV-infected subject, an AIDS patient, atransplant recipient, a pediatric subject, and a pregnant subject. Insome embodiments, the subject has been exposed to HCMV infection. Insome embodiments, the subject is a human.

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

Linker: As used herein, the term “linker” refers to, e.g., in a fusionprotein, an amino acid sequence of an appropriate length other than thatappearing at a particular position in the natural protein and isgenerally designed to be flexible and/or to interpose a structure, suchas an a-helix, between two protein moieties. In general, a linker allowstwo or more domains of a fusion protein to retain 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more of the biological activity of eachof the domains. A linker may also referred to as a spacer.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into an oligonucleotide chain. In some embodiments, anucleic acid is a compound and/or substance that is or can beincorporated into an oligonucleotide chain via a phosphodiester linkage.In some embodiments, “nucleic acid” refers to individual nucleic acidresidues (e.g., nucleotides and/or nucleosides). In some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising individualnucleic acid residues. As used herein, the terms “oligonucleotide” and“polynucleotide” can be used interchangeably. In some embodiments,“nucleic acid” encompasses RNA as well as single and/or double-strandedDNA and/or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,”and/or similar terms include nucleic acid analogs, i.e., analogs havingother than a phosphodiester backbone. For example, the so-called“peptide nucleic acids,” which are known in the art and have peptidebonds instead of phosphodiester bonds in the backbone, are consideredwithin the scope of the present disclosure. The term “nucleotidesequence encoding an amino acid sequence” includes all nucleotidesequences that are degenerate versions of each other and/or encode thesame amino acid sequence. Nucleotide sequences that encode proteinsand/or RNA may include introns. Nucleic acids can be purified fromnatural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, nucleic acids cancomprise nucleoside analogs such as analogs having chemically modifiedbases or sugars, backbone modifications, etc. A nucleic acid sequence ispresented in the 5′ to 3′ direction unless otherwise indicated. The term“nucleic acid segment” is used herein to refer to a nucleic acidsequence that is a portion of a longer nucleic acid sequence. In manyembodiments, a nucleic acid segment comprises at least 3, 4, 5, 6, 7, 8,9, 10, or more residues. In some embodiments, a nucleic acid is orcomprises natural nucleosides (e.g., adenosine, thymidine, guanosine,cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemicallymodified bases; biologically modified bases (e.g., methylated bases);intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages). In someembodiments, the present disclosure is specifically directed to“unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotidesand residues, including nucleotides and/or nucleosides) that have notbeen chemically modified in order to facilitate or achieve delivery.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids., and/or ashaving “polar” or “non-polar” side chains Substitution of one amino acidfor another of the same type may often be considered a “homologous”substitution.

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997; Baxevanis et al., Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more of their corresponding residues are identical over arelevant stretch of residues. In some embodiments, the relevant stretchis a complete sequence. In some embodiments, the relevant stretch is atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500 or more residues.

Suffering from: An individual who is “suffering from” a disease,disorder, or condition (e.g., HCMV infection) has been diagnosed withand/or exhibits one or more symptoms of the disease, disorder, orcondition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, or condition (e.g., HCMV infection) is at risk for developingthe disease, disorder, or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, or condition does not displayany symptoms of the disease, disorder, or condition. In someembodiments, an individual who is susceptible to a disease, disorder, orcondition has not been diagnosed with the disease, disorder, and/orcondition. In some embodiments, an individual who is susceptible to adisease, disorder, or condition is an individual who has been exposed toconditions associated with development of the disease, disorder, orcondition (e.g., the individual has been exposed to HCMV).

Symptoms are reduced: According to the present disclosure, “symptoms arereduced” when one or more symptoms of a particular disease, disorder orcondition is reduced in magnitude (e.g., intensity, severity, etc.) orfrequency. For purposes of clarity, a delay in the onset of a particularsymptom is considered one form of reducing the frequency of thatsymptom. It is not intended that the present disclosure be limited onlyto cases where the symptoms are eliminated. The present disclosurespecifically contemplates treatment such that one or more symptomsis/are reduced (and the condition of the subject is thereby “improved”),albeit not completely eliminated.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount sufficient toconfer a therapeutic effect on the treated subject, at a reasonablebenefit/risk ratio applicable to any medical treatment. The therapeuticeffect may be objective (i.e., measurable by some test or marker) orsubjective (i.e., subject gives an indication of or feels an effect). Inparticular, the “therapeutically effective amount” refers to an amountof a therapeutic protein or composition effective to treat, ameliorate,or prevent a desired disease or condition, or to exhibit a detectabletherapeutic or preventative effect, such as by ameliorating symptomsassociated with the disease, preventing or delaying the onset of thedisease, and/or also lessening the severity or frequency of symptoms ofthe disease. A therapeutically effective amount is commonly administeredin a dosing regimen that may comprise multiple unit doses. For anyparticular immunogenic composition, a therapeutically effective amount(and/or an appropriate unit dose within an effective dosing regimen) mayvary, for example, depending on route of administration, on combinationwith other pharmaceutical agents. Also, the specific therapeuticallyeffective amount (and/or unit dose) for any particular patient maydepend upon a variety of factors including the disorder being treatedand the severity of the disorder, the activity of the specificpharmaceutical agent employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and/or rate of excretion ormetabolism of the specific immunogenic composition employed; theduration of the treatment; and like factors as is well known in themedical arts.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of an immunogenic compositionthat partially or completely alleviates, ameliorates, relieves,inhibits, delays onset of, reduces severity of and/or reduces incidenceof one or more symptoms or features of a particular disease, disorder,and/or condition (e.g., HCMV infection) or the predisposition toward thedisease. Such treatment may be of a subject who does not exhibit signsof the relevant disease, disorder and/or condition and/or of a subjectwho exhibits only early signs of the disease, disorder, and/orcondition. Alternatively or additionally, such treatment may be of asubject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In certain embodiments, the term“treating” refers to the vaccination of a patient.

Tropism: As used herein, the terms “tropism” or “host tropism” or “celltropism” in the context of viruses and other pathogens generally referto the ability of the virus or pathogen to infect a particular celltype. Tropism may refer to a way in which the virus or pathogen hasevolved to preferentially target specific host species or specific celltypes within those species. For example, HCMV can typically infect aremarkably broad cell range within its host, including parenchymalcells, connective tissue cells of virtually any organ and varioushematopoietic cell types. Epithelial cells, endothelial cells,fibroblasts and smooth muscle cells are predominant targets for virusreplication. However, the tropism for various cells varies greatly amongdifferent HCMV strains, e.g., from alterations within the UL128-131 genelocus. In some embodiments, an HCMV strain is able to infectfibroblasts, but not epithelial and/or endothelial cells. In someembodiments, an HCMV strain is able to infect fibroblasts, epithelialcells and endothelial cells.

Vaccination: As used herein, the term “vaccination” refers to theadministration of a composition intended to generate an immune response,for example to a disease-causing agent (e.g., HCMV). For the purposes ofthe present disclosure, vaccination can be administered before, during,and/or after exposure to a disease-causing agent, and in certainembodiments, before, during, and/or shortly after exposure to the agent.In some embodiments, vaccination includes multiple administrations,appropriately spaced in time, of a vaccinating composition.

Vector: As used herein, “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it is associated.In some embodiments, vectors are capable of extra-chromosomalreplication and/or expression of nucleic acids to which they are linkedin a host cell such as a eukaryotic and/or prokaryotic cell. Vectorscapable of directing the expression of operatively linked genes arereferred to herein as “expression vectors.”

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Among other things, the present disclosure provides methods useful fordetermining levels of HCMV infection in host cells and, by extension,determining levels of neutralizing antibodies present in a sample. Thepresent disclosure encompasses the recognition that HCMV viruses thathave a fluorescent moiety permit detection of viral infection (e.g., byassessing fluorescence in cells after contacting the host cell with thevirus). In some embodiments, levels of HCMV infection are determined byfluorescence detection where the virus has been preincubated with a testsample (e.g., a serum sample) from a subject. In some embodiments, thesubject has been administered a candidate HCMV vaccine.

I. HCMV Infection and Vaccines

Human cytomegalovirus (HCMV), a β-herpesvirus, is a ubiquitouslyoccurring pathogen. In general, entry of herpesviruses into cells is acomplex process initiated by adsorption and receptor binding andfollowed by fusion of the virus envelope with a cell membrane. Fusiongenerally occurs at either the plasma membrane or an endosomal membrane.HCMV infects multiple cell types in vivo, including epithelial cells,endothelial cells and fibroblasts (Plachter B et al., 1996 Adv Virus Res46:195-261). It fuses with the plasma membranes of fibroblasts (ComptonT et al., 1992 Virology 191:387-395), but enters retinal pigmentedepithelial cells and umbilical vein endothelial cells via endocytosis(Bodaghi B et al., 1999 J Immunol 162:957-964; Ryckman B J et al., 2006J Virol 80:710-722). The mechanism by which herpesviruses choose theirroute of entry remains unclear. It is generally assumed that entrypathways are mainly determined by the host cell, but there is evidencefor tropic roles of virion glycoproteins (Wang X et al., 1998 J Virol72:5552-5558). HCMV encodes two gH/gL complexes: gH/gL/gO andgH/gL/UL128/UL130/UL131. The gO-containing complex is sufficient forfibroblast infection, whereas the pUL 128/UL130/UL131-containing complexis important for HCMV infection of endothelial and epithelial cells. Asused herein, the terms “tropism” or “host tropism” or “cell tropism” inthe context of viruses and other pathogens generally refer to theability of the virus or pathogen to infect a particular cell type.Tropism may refer to a way in which the virus or pathogen has evolved topreferentially target specific host species or specific cell typeswithin those species. In some embodiments, an HCMV strain is able toinfect fibroblasts, but not epithelial and/or endothelial cells. In someembodiments, an HCMV strain is able to infect fibroblasts, epithelialcells and endothelial cells.

HCMV infects 50-85% of adults by 40 years of age (Gershon A A et al.,1997 in Viral Infections ofHumans, 4^(th) edition, New York; PlenumPress:229-251). Most healthy individuals who acquire HCMV after birthdevelop few, if any, symptoms. However, HCMV disease is the cause ofsignificant morbidity and mortality in immunocompromised individuals,such as recipients of hematopoietic cell transplants (HCT) andsolid-organ transplants (SOT) (Pass R F 2001 Cytomegalovirus. In FieldsVirology. 4^(th) edition, Philadelphia; Lippincott Williams &Wilkens:2675-2705). In SOT or HCT populations, HCMV disease can occureither from new infection transmitted from the donor organ or HCT, orcan recur as a result of reactivation of latent virus in the recipient.In HIV-infected individuals, HCMV infection accelerates progression toAIDS and death, despite availability of antiretroviral therapy (DeaytonJ R et al., 2004 Lancet 363:2116-2121). In addition in the US, HCMV isthe most common intrauterine infection and causes congenitalabnormalities resulting in death or severe birth defects, includingdeafness and mental retardation, in approximately 8,000 infants eachyear (Stagon S et al., 1986 JAMA 256:1904-1908).

Immune responses which control HCMV are incompletely understood. Byanalogy to other human herpesviruses it can be assumed that bothcellular and humoral immune responses play an important role (Kohl S1992 Current topics in Microbiology and Immunology 179:75-88). Formurine CMV it was shown that either a cytotoxic T cell response or thepassive transfer of neutralizing antibodies is sufficient to protectagainst a lethal challenge (Rapp M et al., 1993 MultidisciplinaryApproach to Understanding Cytomegalovirus Disease 327-332; Reddehase M Jet al., 198 J Virology 61:3102-3108).

Control of HCMV in immunocompromised persons is primarily associatedwith cellular immune responses; both CD8⁺ and CD4⁺ T lymphocytes appearto be important for protection against CMV disease (Gamadia L E et al.,2003 Blood 101:2686-2692; Cobbold M et al., 2005 J Exp Med 202:379-386).The cellular immune response to CMV includes CD4⁺ helper T-lymphocyteand CD8⁺ Cytotoxic T-lymphocyte responses to a number of antigens, foundin the viral tegument, the region of the viral particle between theenvelope and capsid. A recent study of CMV-specific CD4⁺ and CD8⁺ Tcells from healthy donors used overlapping peptides from a series of CMVopen reading frames to identify antigens recognized after CMV infection(Sylwester A W et al., 2005 J Exp Med 202:673-685). The CMV tegumentphosphoprotein 65 (pp65) and surface glycoprotein gB were the antigensmost frequently recognized by CD4⁺ T cells, and pp65 was also one of theantigens most frequently recognized by CD8⁺ T cells.

In contrast to the transplant setting, the maternal humoral immuneresponse against the virus seems to be important in preventing HCMVdisease in the newborn. Antibodies to surface glycoproteins, especiallygB, appear to be critical for protection against the maternal-fetaltransfer of HCMV (Fowler K B et al., 2003 JAMA 289:1008-1011). Moreover,in an earlier vaccination study it was shown that protection fromre-infection is correlated with neutralizing antibodies (Adler S P etal., 1995 J Infectious Diseases 171:26-32). The humoral immune responseto HCMV is dominated by responses to viral envelope glycoproteinspresent in the outer envelope of the virus particle (e.g., gB and gH).

In the case of HCMV, direct evaluation of immunological effectorfunctions is difficult since the virus is strictly species specific andno animal model system is available. However, murine CMV and guinea pigCMV have been used to evaluate vaccine strategies in these host species.

A CMV vaccine that induces both protective T cell and neutralizingantibody responses has the potential to prevent infection or ameliorateCMV disease due to congenital infection or transplantation.

The first live, attenuated HCMV vaccine candidate tested in humans wasbased on the laboratory-adapted AD169 strain. Subsequent trials withanother laboratory-adapted clinical isolate, the Towne strain, confirmedthat live attenuated vaccines could elicit neutralizing antibodies, aswell as CD4+ and CD8+T lymphocyte responses. The efficacy of the Townevaccine was assessed in a series of studies in renal transplantrecipients. Although the Towne vaccine did provide a protective impacton HCMV disease it failed to prevent HCMV infection aftertransplantation (Plotkin S A et al., 1984 Lancet 1:528-530). Townevaccine was also evaluated in a placebo-controlled study of seronegativemothers who had children attending group daycare where it failed toprevent these women from acquiring infection from their HCMV-infectedchildren (Adler S P et al., 1995 J Infectious Diseases 171:26-32). Aninterpretation of these studies was that the Towne vaccine wasoverattenuated. To explore this possibility a series of geneticrecombinants in which regions of the unattenuated “Toledo” strain of CMVwere substituted for the corresponding regions of the Towne genome,resulting in the construction of Towne/Toledo “chimeras” that containsome, but not all, of the mutations that contribute to the Towne vaccineattenuation (Heineman T C et al. 2006 J Infect Disease 193:1350-1360).The safety and tolerability of four Towne/Toledo “chimeras” is beingtested in a Phase I trial. Long-term safety concerns about the potentialrisk of establishing a latent HCMV infection have hindered the furtherdevelopment of live, attenuated vaccines.

The leading subunit CMV vaccine candidate is based on the envelopeglycoprotein, gB, (purified recombinant gB vaccine is manufactured bySanofi-Pasteur Vaccines) due to this protein's ability to elicithigh-titer, virus-neutralizing antibody responses during naturalinfection. The recombinant gB vaccine elicits neutralizing antibodyresponses and has an excellent safety profile, however, it excludesother glycoprotein targets of neutralizing antibody response and moreimportantly T-lymphocyte targets. The vaccine requires MF59 adjuvant tooptimize immunogenicity. In the most recent trial, this vaccine providedan overall 50% efficacy for prevention of CMV infection in a Phase 2clinical trial in young women (Pass R F et al., 2009 N Engl J Med360:1191-1199). Other viral proteins being evaluated as subunit vaccinecandidates include pp65 and IE1, both of which elicit T-cell responses.

DNA vaccines elicit robust cellular and humoral immune responses inanimals and are well suited to specificity and precision in vaccinedesign. DNA vaccines have been developed for CMV and have focused on gB,IE1 and pp65 proteins as the candidate target immunogens. A bivalent CMVDNA vaccine candidate (Wloch M K, 2008 J Infectious Diseases297:1634-1642), using plasmid DNA encoding pp65 and gB and a trivalentvaccine candidate (Jacobson M A, 2009 Vaccine 27:1540-1548) that alsoincludes a third plasmid encoding the IE1 gene product have beendeveloped by Vical Vaccines (U.S. Pat. No. 7,410,795). The trivalent DNAvaccine alone had minimal immunogenicity irrespective of route ofadministration. However the CMV DNA vaccine did appear to safely primefor a memory response to CMV antigens observed after administration of alive, attenuated CMV (Towne).

In a vectored vaccine approach, the gene product of interest isexpressed in the context of a non-replicating (usually viral) carrier.One example of this is a canarypox vector called ALVAC developed byVirogenetics and Sanofi-Pasteur Vaccines, which is an attenuatedpoxvirus that replicates abortively in mammalian cells. ALVAC expressingCMV gB and ALVAC expressing pp65 (U.S. Pat. No. 6,267,965) have beentested in clinical trials. ALVAC-CMV(gB) did not induce neutralizingantibodies but did prime for higher neutralizing antibody titers aftersubsequent infection with the Towne strain CMV (Adler S P et al., 1999 JInfectious Diseases 180:843-846), although it did not appear to boostneutralizing antibody titers after subsequent immunization with gBsubunit/MF59 vaccine (Bernstein D I et al., 2002 J Infectious Diseases185:686-690). A canarypox vector expressing pp65, ALVAC-CMV(pp64),induced long-lasting CTL responses in all originally seroncgativevolunteers, at frequencies comparable to naturally seropositiveindividuals (Berencsi K et al., 2001 J Infectious Diseases183:1171-1179). Another approach used to express gB as a vectoredvaccine is the use of an alphavirus replicon system by AlphaVax Inc(U.S. Pat. No. 7,419,674). This approach involves apropagation-defective single-cycle RNA replicon vector system derivedfrom an attenuated strain of an alphavirus, Venezuelan EquineEncephalitis (VEE) virus, to produce virus-like replicon particles(VRPs) expressing pp65, IE1 or gB protein (Berstein et al., 2010 Vaccine28:484-493). A two component alphavirus replicon vaccine was used toexpress the three CMV proteins as a soluble form of CMV gB (Townestrain) and a pp65/IE1 fusion protein (Reap E A et al., 2007 Vaccine25:7441-7449) was found to be safe and induced high levels ofneutralizing antibody and polyfunctional CD4+ and CD8+ antigen-specificT cell responses. The Geometric Mean Titre (GMT) for the high dose groupwas about half the GMT in 12 naturally infected, CMV seropositiveindividuals tested in the assay.

A candidate for vaccination against HCMV currently in preclinicaldevelopment is the “dense body” vaccine. Dense bodies (DBs) areenveloped, replication-defective particles formed during the replicationof CMVs in cell culture. They contain both envelope glycoproteins andlarge quantities of pp65 protein. DBs are non-infectious and immunogenicbut incapable of establishing latent HCMV infection in the vaccinerecipient. DBs have been shown to be capable of inducing virusneutralizing antibodies and T-cell responses in mice in the absence ofviral gene expression (Pepperl S et al., 2000 J Virol 74:6132-6146, PCTPublication No. WO 00/53729 and U.S. Pat. No. 6,713,070).

Additional candidates contemplated for vaccination against HCMV arevirus like particles (VLPs). Retroviruses are enveloped RNA viruses thatbelong to the family Retoviridae. After infection of a host cell by aretrovirus, RNA is transcribed into DNA via the enzyme reversetranscriptase. DNA is then incorporated into the host cell's genome byan integrase enzyme and thereafter replicates as part of the host cell'sDNA. The Retroviridae family includes the following genusAlpharetrovirus, Betaretrovirus, Gammearetrovirus, Deltaretrovirus,Epsilonretrovirus, Lentivirus and Spumavirus. The hosts for this familyof retroviruses generally are vertebrates. Retroviruses produce aninfectious virion containing a spherical nucleocapsid (the viral genomein complex with viral structural proteins) surrounded by a lipid bilayerderived from the host cell membrane.

Retroviral vectors can be used to generate enveloped virions that areinfectious and either replication-competent or replication-defective.Replication-competent infectious retroviral vectors contain all of thenecessary genes for virion synthesis and continue to propagatethemselves once infection of the host cell occurs. Replication-defectiveinfectious retroviral vectors do not spread after the initial infection.This is accomplished by replacement of most of the coding regions of theretrovirus with genes or nucleotide sequences to be transferred; so thatthe vector is incapable of making proteins required for additionalrounds of replication.

Alternatively or additionally, retroviral vectors can be used togenerate virus-like particles (VLPs) that lack a retrovirus-derivedgenome and are both non-infectious and non-replicating. Because of VLPsadvantageous properties, VLPs may be utilized as an antigen deliverysystem. Furthermore, because VLPs are non-infectious, they can beadministered safely as an immunogenic composition (e.g., a vaccine).VLPs are generally structurally similar to enveloped virions describedabove, but lack a retrovirus-derived genome, making it unlikely thatviral replication will occur. Expression of capsid proteins (e.g., Gag)of some viruses (e.g., murine leukemia viruses, such as Moloney Murineleukemia virus (MMLV)) leads to self-assembly into particles similar tothe corresponding native virus, which particles are free of viralgenetic material.

A wide variety of VLPs have been prepared. For example, VLPs includingsingle or multiple capsid proteins either with or without envelopeproteins and/or surface glycoproteins have been prepared. In some cases,VLPs are non-enveloped and assemble by expression of just one majorcapsid protein, as shown for VLPs prepared from hepadnaviruses (e.g.,Engerix™, GSK and Recombivax HB™, Merck), papillomaviruses (e.g.,Cervarix™, GSK and Gardasil™, Merck), paroviruses, or polyomaviruses. Insome embodiments, VLPs are enveloped and can comprise multiple antigenicproteins found in the corresponding native virus. VLPs typicallyresemble their corresponding native virus and can be multivalentparticulate structures. In some embodiments, antigenic proteins may bepresented internally within the VLP, as a component of the VLPstructure, and/or on the surface of the VLP. In some embodiments,presentation of an antigen in the context of a VLP is advantageous forinduction of neutralizing antibodies against the antigen as compared toother forms of antigen presentation, e.g., soluble antigens notassociated with a VLP. Neutralizing antibodies most often recognizetertiary or quaternary structures; this often requires presentingantigenic proteins, like envelope glycoproteins, in their native viralconformation. Alternatively or additionally, VLPs may be useful forpresenting antigens in a context which induces cellular immunity (e.g.,T cell response). In some embodiments, use of antigen combinations inVLP systems can generate improved immune response.

II. Detectable HCMV

As described above, among other things, the present disclosure providesmethods for determining levels of HCMV infection in host cells and, byextension, determining levels of neutralizing antibodies present in asample. The present disclosure encompasses the recognition that HCMVviruses that have a fluorescent moiety permit detection of viralinfection (e.g., by assessing fluorescence in cells after contacting thehost cell with the virus). In some embodiments, levels of HCMV infectionare determined by fluorescence detection where the virus has beenpreincubated with a test sample (e.g., a serum sample) from a subject.In some embodiments, the subject has been administered a candidate HCMVvaccine.

Provided methods utilize an HCMV virus that includes a fluorescentmoiety. Any HCMV virus capable of infecting a host cell described hereincan be engineered to include a fluorescent moiety. In some embodiments,to infect fibroblasts, an HCMV virus that includes all or a portion of agH/gL/gO complex can be engineered to include a fluorescent moiety. Insome embodiments, to infect endothelial cells and/or epithelial cells,an HCMV virus that includes all or a portion of a gH/gL/UL128/UL130UL131complex can be engineered to include a fluorescent moiety. Modified HCMVstrains that are amenable to fluorescent detection are known in the artand may be used in accordance with the present disclosure. For example,UL32-EGFP-HCMV-TB40 is an in vitro recombination of HCMV strain TB40with a plasmid carrying the TB40 UL32 gene fused to GFP (ATCC; VR-1578).The UL32-EGFP-HCMV-TB40 recombinant strain gives rise to a recombinantHCMV virus with GFP fused to the C terminus of the tegumentphosphoprotein pp150, the product of the UL32 gene (Sampaio et al., 2005Journal of Virology 79(5):2754). Because GFP is associated with a viralstructural protein, virus particles fluoresce green under appropriateillumination. The UL32-EGFP-HCMV-TB40 strain has been demonstrated tohave tropism for fibroblast cells (Sampaio et al., 2005 Journal ofVirology 79(5):2754). An additional HCMV strain that is amenable tofluorescent detection is HB 15-t178b, which contains the CMV strainAD169 genome and a GFP reporter cassette (Saccoccio et al., 2011 Vaccine29(15):2705).

It is to be understood that the term fluorescence, as used herein,refers to a moiety that luminesces. Typically fluorescent moietiescontain electrons which can absorb electromagnetic energy at onewavelength and emit electromagnetic energy at a second wavelength. Someproteins or small molecules in cells are naturally fluorescent (e.g.,NADH, tryptophan, endogenous chlorophyll, phycoerythrin, or greenfluorescent protein (GFP)). It will be appreciated that various mutantsof fluorescent proteins have been engineered and may be used inaccordance with the present disclosure, such as EGFP, blue fluorescentprotein (e.g., EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein(e.g., ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g., YFP,Citrine, Venus, YPet), redox sensitive GFP (e.g., roGFP), and monomericGFP, among others. GFP and other fluorescent proteins can be expressedexogenously in cells alone or as a fusion protein. This approach permitsfluorescent proteins to be used as reporters for any number ofbiological events, such as subcellular localization and expressionpatterns.

Alternatively or additionally, specific or general proteins, nucleicacids, lipids or small molecules can be labeled with an extrinsicfluorophore, a fluorescent dye which can be a small molecule, protein orquantum dot. Exemplary fluorophores include, but are not limited to, 1,5IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA);5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine);6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin;7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-I methylcoumarin;9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); AFPs—AutoFluorescent Protein—(QuantumBiotechnologies) see sgGFP, sgBFP; Alexa Fluor® 350; Alexa Fluor® 430;Alexa Fluor® 488; Alexa Fluor® 532; Alexa Fluor® 546; Alexa Fluor® 568;Alexa Fluor® 594; Alexa Fluor® 633; Alexa Fluor® 647; Alexa Fluor® 660;Alexa Fluor® 680; Alizarin Complexon; Alizarin Red; Allophycocyanin(APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; AminoactinomycinD; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC;APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B;Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ;Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue FluorescentProtein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst);bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy492/515;Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FLATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-Xconjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE;BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein;Calcein Blue; Calcium Crimson™; Calcium Green™; Calcium Green™-1 Ca²⁺Dye; Calcium Green™-2 Ca²⁺; Calcium Green™-5N Ca²⁺; Calcium Green™-C18Ca²⁺; Calcium Orange™; Calcofluor White; Carboxy-X-rhodamine (5-ROX);Cascade Blue®); Cascade Yellow™; Catecholamine; CCF2 (GeneBlazer); CFDA;CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll; ChromomycinA; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; CoumarinPhalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan;Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP;cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; DansylCadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3′DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD-Lipophilic Tracer; DiD (Di1C18(5)); DIDS; Dihydorhodamine123 (DHR); Dil (Di1C18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(Di1C18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS;DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight;Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline);FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3;Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald;Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM® 1-43; FM®4-64; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer, (CCF2); GFP (S65T); GFP red shifted(rsGFP); GFP wild type′ non-UV excitation (wtGFP); GFP wild type, UVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;Calcein/Ethidium homodimer, LOLO-1; LO-PRO-1; Lucifer Yellow; LysoTracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso TrackerRed; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensorYellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red;Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange;Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; MaxilonBrilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker GreenFM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane;Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green PyronineStilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline;Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; OregonGreen®; Oregon Green® 488; Oregon Green® 500; Oregon Green® 514; PacificBlue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-1 PRO-3; Primuline;Procion Yellow; Propidium lodid (P1); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T;Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™(super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; StilbeneIsothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein;SNARF 1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange;Spectrum Red; SPQ (6-methoxy-N-(3 sulfopropyl)quinolinium); Stilbene;Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOXGreen; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); TexasRed®; Texas Red®-X conjugate; Thiadicarbocyanine (DiSC3); Thiazine RedR; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3; YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductornanoparticles such as quantum dots; or caged fluorophore (which can beactivated with light or other electromagnetic energy source), or acombination thereof.

III. Infection Assays

Determination of infectious titer of HCMV typically includes contactinga host cell that is susceptible to infection by HCMV with serialdilutions of the virus (e.g., HCMV that includes a fluorescent moiety),under conditions that allow cell infection in the absence of any testsubstance. The number of target cells expressing a reporter geneconstruct (e.g., a fluorescent moiety, e.g., GFP) may be determined(e.g., by flow cytometry) to calculate the infectious titer of the viruspreparation.

To assess the presence and/or activity of neutralizing antibodies inserum of a subject to whom a candidate vaccine has been administered,the serum may be pre-incubated with HCMV (e.g., HCMV that includes afluorescent moiety, e.g., GFP) for a period of time sufficient forneutralizing antibodies to reduce infectivity of the HCMV (e.g., atleast 15 minutes, at least 30 minutes, at least 1 hour, at least 2hours, or more). Serial dilutions of the pre-incubated mixture of serumand HCMV (e.g., HCMV that includes a fluorescent moiety) may then beused to contact a host cell that is susceptible to infection by HCMVunder conditions that allow infection. A person of ordinary skill in theart will be able to determine appropriate dilutions of serum forinfection assays. For example, in some embodiments, dilutions tested are1:6, 1:12, 1:24, 1:48, 1:96, 1:192, or combinations thereof.

It will be appreciated that conditions that allow infection may varydepending on a variety of factors, including viral strain, host celltype, temperature, cell confluence, viral concentration, among others.One of ordinary skill in the art would be able to modify infectionconditions appropriately. In some embodiments, infection conditionsinclude incubation of the host cell with virus for at least 1 hour, atleast 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, atleast 6 hours, at least 7 hours, at least 8 hours, or more. It will alsobe appreciated that monitoring for infection of cells may include visualcellular morphology assessment (e.g., for cellular swelling androunding) and/or visual fluorescence assessment (e.g., detectingfluorescence on a device such as a fluorescence microscope). Afterinfection, host cells may be collected (e.g., by washing andtrypsinizing and/or scraping) and analyzed by fluorescent detectionmethods available in the art and described herein.

Any host cell susceptible to infection by HCMV can be used in themethods described herein. Exemplary host cells include, but are notlimited to human fibroblast cells, such as human foreskin fibroblasts(HFF), and human epithelial cells such, as retinal pigmented epithelialcells (ARPE-19). Medium in which host cells are grown during theinfection assay may vary with cell type. For example, in someembodiments, HFF infection medium includes MEM+5% FBS+1% PenStrep. Insome embodiments, APRE infection medium includes DMEM:F-12+1% FBS.

IV. Fluorescence Detection and Neutralizing Antibody Assessment

Fluorescence may be detected using any appropriate method, including,for example, flow cytometry analysis, fluorescent activated cellsorting, or flow microfluorometry. It will be appreciated that thesensitivity of fluorescence detection generally depends on the number ofcopies of the fluorescent entity in the detection system, the efficiencyof the detection instrument, and the fluorescence brightness of thefluorescent entity relative to background fluorescence that arises fromendogenous biological fluorescent entities in the sample and fromnon-specific association of the fluorescent entity with the sample. Thebrightness of the fluorescent entity, in turn, depends on the quantumefficiency of the fluorescent entity that produces the fluorescencesignal and the light absorbing capability (quantified by the extinctioncoefficient) of the fluorescent entity.

It is to be understood that cells can be identified and/or isolatedbased on levels of surface and/or intracellular fluorescence usingreporter molecules such as green fluorescent protein (GFP). Generally, acorrelation between fluorescence intensity and protein production (e.g.,viral infection and production) will be observed. For example, in someembodiments, high levels of fluorescence intensity in a host cellcorrelates with high level of viral infection of the host cell.

In some embodiments, cells are assessed for overall fluorescence level(e.g., irrespective of subcellular localization of the fluorescence). Insome embodiments, cells are assessed for fluorescence level in aparticular subcellular location (e.g., the nucleus) of the cell. Ingeneral, flow cytometry permits quantitative phenotyping of largenumbers of cells, however, many flow cytometry applications do notinclude an ability to image cells as they are quantitated. In someembodiments, detection and quantitation of fluorescence of cells issufficient for assessing infection. It will be appreciated that in somecases it is desirable to determine the localization of fluorescencewithin the cell. In some embodiments, flow cytometry analysis of cellsmay be combined with visual assessment of fluorescence. Dual analysis offluorescence level and localization may be performed using multipledevices (e.g., flow cytometry and fluorescence microscopy) or on asingle device. Such devices that permit fluorescence analysis and/orquantitation in conjunction with localization analysis are available inthe art. For example, ImageStream^(X) (Amnis®) quantifies both intensityand localization of fluorescence and permits imaging of more than 50,000cells per minute.

Cell fluorescence levels may be quantitated by any appropriate methodknown in the art. Cell fluorescence levels may be compared to areference level. In some embodiments, the reference level is apredetermined or historical reference level. In some embodiments, thereference level is obtained by side-by-side comparison with a referencesample (e.g., a positive and/or negative control).

The advent of screening and selection methods that use flow cytometryand cell sorting considerably increase the number of cells that can bescreened. For example, several million cells can be screened in a shorttime, and subpopulations and single cells can be isolated from withinmixed-cell populations even when they are present at frequencies as lowas 10⁻⁶ within the population. It will be appreciated that one of theadvantages of provided methods is that sample assessment can be achievedin a high-throughput manner. In some embodiments, provided methodsincrease throughput by 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more ascompared to known infection and/or neutralizing antibody detectionmethods (e.g., staining of viral proteins, fluorescence microscopy,ELISPOT, etc.). Provided methods may be performed in a multi-well plateformat and are therefore particularly suitable for use in mid-to-highthroughput screening. In some embodiments, the multi-well plates have 96wells. In some embodiments, the multi-well plates have another number ofwells, which include but is not limited to plates with 6, 12, 24, 384,864 or 1536 wells. The terms “multi-well plate” and “microtiter plate”are used interchangeably.

In some embodiments, live cells are analyzed. In some embodiments, cellsare fixed prior to analysis.

In some embodiments, the present disclosure provides methods formeasuring anti-HCMV neutralizing antibodies by correlating fluorescencedetection and/or viral infection of host cells. For example, an HCMVthat includes a fluorescent moiety (e.g., GFP) may be pre-incubated withserum from a subject immunized with an HCMV candidate vaccine.Neutralizing antibodies present in the serum will reduce HCMV (e.g.,HCMV that includes a fluorescent moiety) from infecting a host cell thatis normally susceptible to infection by HCMV. The pre-incubated mixtureincluding HCMV (e.g., HCMV that includes a fluorescent moiety) and serumcan then be used to contact such a host cell and fluorescence levels ofthe host cell may be assessed. Based on fluorescence levels in the hostcell, a level of infection of the host cell can be determined. Level ofinfection of the host cell generally is inversely related to thepresence and amount neutralizing antibody in the serum, which in turn,correlates with efficacy of the candidate vaccine to elicit atherapeutic response (e.g., a protective immune response). For example,the more efficacious a candidate vaccine at inducing neutralizingantibodies in a subject, the more neutralizing antibodies will bepresent in the serum, in turn corresponding to a decrease in ability ofan HCMV that includes a fluorescent moiety to infect a host cell afterpre-incubation with the serum, further corresponding to a decrease influorescence detection in the host cell after contacting it with an HCMVthat includes a fluorescent moiety. In some embodiments, methods of thedisclosure further include selecting and/or identifying, based on acorrelation described herein, a candidate vaccine (e.g., an HCMVcandidate vaccine) as a vaccine that induces neutralizing antibodies.

EXAMPLES

The following examples describe some exemplary modes of making andpracticing certain compositions that are described herein. It should beunderstood that these examples are for illustrative purposes only andare not meant to limit the scope of the compositions and methodsdescribed herein.

Example 1: Immunization of Rabbits with Virus-Like Particles

This Example describes exemplary immunization of rabbits with virus-likeparticles containing various recombinant HCMV antigens.

HEK 293T cells (ATCC, CRL-11268) were transiently transfected usingcalcium phosphate methods with expression plasmids encoding variousrecombinant HCMV antigens. Expression of various HCMV antigens by theHEK 293 cells was confirmed by flow cytometry. After 48 to 72 hours oftransfection, supernatants containing the VLPs were harvested andfiltered through 0.45 μm pore size membranes and further concentratedand purified by ultracentrifugation through a 20% sucrose cushion in aSW32 Beckman rotor (25,000 rpm, 2 hours, 4° C.). Pellets wereresuspended in sterile endotoxin-free PBS (GIBCO) to obtain 500 timesconcentrated VLP stocks. Total protein was determined on an aliquot by aBradford assay quantification kit (BioRad). Purified VLPs were stored at−80° C. until used.

Rabbits were immunized intramuscularly at t=0 and t=8 weeks with VLPs asshown in Table 1 below. Serum was collected at t=4, 6, 8, 10, 13 and 16weeks.

TABLE 1 Test Article # (n = 6/group) Dose Test Article Description 1 100μg gB/pp65 bivalent VLPs 6 100 μg/each gB/pp65 bivalent VLPs + gH-G/pp65bivalent VLPs (1:1 ratio)

Enzyme-linked Immunosorbent Assay (ELISA) was performed to determinerabbit serum HCMV IgG content. FIG. 1 shows potent and sustainedimmunity in rabbits immunized with gB/pp65 bivalent VLPs (upper) andgB/pp65 bivalent VLPs+gH-G/pp65 bivalent VLPs (lower).

Example 2: Flow Cytometry-Based Neutralization Activity

This Example describes assessment of neutralizing antibody responses toHCMV in serum from immunized animals. The objectives of this Exampleincluded, but were not limited to: 1) confirming the immunogenicity ofCMV clinical candidate components selected from other animal studies; 2)assessing dose and potential synergy and/or antagonism withadministration routes (e.g., IM vs. IP administration); and 3) assessingimmunological boosting and durability of immunity.

Serum samples from rabbits immunized with Test Article #6 from Example 1were collected at t=0 (POV) and t=2 weeks (P2Vd14) time points. Pooled,heat inactivated (HI) samples from this group were then tested forneutralizing antibody activity as described below.

Serum samples were diluted ⅙ to 1/96. HFF cells (P=9, used 24 hours postseeded) and TB40-230212 freshly harvested virus were used. TB40-230212virus is descendant of TB40-010212. TB40-010212 was obtained from ATCC(ATCC VR-1578; Human herpesvirus 5; UL32-EGFP-HCMV-TB40). Virusinfection duration was 15 days in a T150 infection flask (Table 2).Microscopic observations indicated the virus was infective. Infectionmedium was TB40-HFF-1 infection medium (MEM+5% FBS+1% Pen/Strep) or VR1814-APRE-19 infection medium (DMEM:F-12+1% FBS).

TABLE 2 Virus infection Infection Microscope Virus Lot# AscendantDuration flask observation TB40 230212 TB40-010212 15 days T150infective (new ATCC)

An HFF seeded T150 flask was infected with CMV TB40-010212 (5 vials)+20ml of infectious media (MEM—Minimum Essential Medium Eagle from SigmaCat.No. M4655+5% FBS+1% P/S). After 15 days in CO₂ incubator/37′C, theflask was well-infected. Virus was harvested by removing almost theentire supernatant from the flask and keeping sterile in 50 mL Falcontube. The remaining supernatant, around 5-7 mL, served to help scrapingthe cells with cell scraper (NUNC, Cat#179707). After scraping, strongpipetting up and down (up to 10×) facilitated virus release from cells.Half of the original supernatant volume from Falcon tube was added (cca.8 mL) in order to get more potent virus.

Five vials of TB40-010212 (ascendant) from liquid nitrogen was infectedin one T150 flask, incubated at 37° C., 5% CO₂ for 1 hour. Twelve mL ofinfection media was added after incubation. Virus was concentrated in 15mL of supernatant.

Cytogam™ (CMV-IGIV—Cytomegalovirus Immune Globulin Intravenous—Human;CSL Behring; Commercial concentration 2.5 g/50 ml or 50 mg/ml) was usedas a positive control. A 1/20 dilution was made as a base concentration(1 in 20 dilution: 100 μL of Cytogam™ 50 mg/mL+1900 μL of infectiousmedia) and later used for making dilutions, the same dilutions used forrabbit sera. Cytogam™ was heat inactivated under the same conditions asrabbit sera (as described in Tables 3-5 below)

TABLE 3 Infection Assay Pooled HI P0V ⅙ ½ virus 2 6-well plate wells1^(st) plate 1/12 ½ virus 2 6-well plate wells 1/24 ½ virus 2 6-wellplate wells 1/48 ½ virus 2 6-well plate wells 2^(nd) plate 1/96 ½ virus2 6-well plate wells cells 2 6-well plate wells Pooled HI P2V ⅙ ½ virus2 6-well plate wells 3^(rd) plate 1/12 ½ virus 2 6-well plate wells 1/24½ virus 2 6-well plate wells 1/48 ½ virus 2 6-well plate wells 4^(th)plate 1/96 ½ virus 2 6-well plate wells cells 2 6-well plate wells HICytogam ™ ⅙ ½ virus 2 6-well plate wells 5^(th) plate (base 1/20) 1/12 ½virus 2 6-well plate wells 1/24 ½ virus 2 6-well plate wells 1/48 ½virus 2 6-well plate wells 6^(th) plate 1/96 ½ virus 2 6-well platewells TB40-230212 1 in 2 diluted 2 6-well plate wells

TABLE 4 Pooled sera dilutions for P0V, P2Vd14 and for Cytogam ™ MediaNeat Final without virus conc. FBS to make Sera used used in Sera Usedsera dilutions Total in assay assay vs. dilution Used sera (ul) (ul)(ul) (ul) Virus ⅓ 100 ul 200 300 130 ul ⅓ 130 ⅙ neat sera vs. ½ ⅙ 150 ul⅓ 150 300 130 ul ⅙ 130 1/12 vs. ½ 1/12 150 ul ⅙ 150 300 130 ul 1/12 1301/24 vs. ½ 1/24 150 ul 1/12 150 300 130 ul 1/24 130 1/48 vs. ½ 1/48 150ul 1/24 150 300 130 ul 1/48 130 1/96 vs. ½

TABLE 5 HFF-1 cell growth and infection media Concentration ReagentSupplier Cat# Lot# Exp of components Volume HFF-1 cells DMEM HyCloneSH30243.01 AWH17628 August 2012 80% 420 ml growth (Dulbecco's mediaModified (DMEM + Eagle 15% FBS + Medium) 1% FBS (Fetal HyCloneSH30396.03 AVC67186 March 2015 15% 75 ml Pen/Strep) bovine serum)Pen/Strep Sigma P0781-100 ML 031M0787 October 2012  1% 5 ml (PenicillinStreptomycin Solution) HFF MEM Sigma M4655-500 ml RNC0312 September 201294% 470 ml infection (Minimum media Essential TB40 Medium (MEM + Eagle)5% FBS + FBS (Fetal Hyclone SH30396.03 AVC67186 March 2015  5% 25 ml 1%bovine serum) Pen/Strep) Pen/Strep Sigma P0781-100 ML 031M0787 October2012  1% 5 ml (Penicillin Streptomycin Solution)

Each particular concentration of sera or Cytogam™ and ½ final virusdilution were combined and rotated at 37′C/1 hr. Ready to use 6-wellplate with 95%-100% confluent HFF cells were carefully rinsed twice withwarm PBS, then virus-sera mixture was applied in duplicate as 100 μLmixture/well+200 μL infection media to avoid drying over 4 hrs.Incubation was at 37′C/5% CO₂/4 hrs (rocking was every 60 minutes).After a 4-hour incubation, contents were carefully aspirated from eachwell with a pipette and 3 mL of fresh infectious media were added.Plates were kept in the incubator at 37′C/5% CO₂ for 10 days. Dailychecking of virus infection in cells was performed usually after 5 dayspost-infection. Swelling and rounding cells were visualized by lightmicroscope or green fluorescence detection with a fluorescentmicroscope.

For sample collection, media was aspirated from each well. Each well wasrinsed twice with PBS (HyClone DPBS/modified without Calcium andMagnesium; Cat#SH30028.02) and 100 μL of 1× Trypsin-EDTA (Sigma;Cat#T4174-100 ml) and 100 μL of PBS were added. Samples were kept in CO₂incubator 2-3 minutes until cells were well trypsinized. 1 mL/well ofPBS+5% FBS was added to stop trypsin. Samples were collected intotransparent flow intended tubes (two wells for same sample/tube) (BDFalcon, 5 ml polystyrene round-bottom, REF 352054). Plates were checkedunder the light microscope to confirm all cells were collected (if notadd additional 500 μL PBS+5% FBS was used to collect the rest). Sampleswere spun down at 900 rpm for 10 minutes. Supernatant was discarded andthe pellet kept. The tube was vortexed so that pellet was dispersed inthe leftover buffer. The remaining steps were performed with minimalexposure to light. 200 μL fixative (BD Biosciences, BD Cytofix, Fixationbuffer Cat#554655, 100 ml) was added and incubated on ice for 15minutes. 1 mL/tube of mixture PBS+5% FBS was added to stop Cytofix.Samples were spun down at 900 rpm for 10 minutes. Supernatant wasdiscarded and the pellet kept. The pellet was reconstituted in 500 μL ofPBS+5% FBS and vortexed vigorously before putting on flow cytometer.

Samples were subjected to flow cytometry and analyzed using Cellquestsoftware. FSC and SSC parameters were set to “linear”, while all otherparameters were set to “log.” 100,000 cells were collected during flowcytometric analysis of infected cells. Usual conditions for flow usingHFF cells and TB40 CMV were FSC E-1 and 4.83; SSC 325; FL1 427, althoughparameters can be slightly changed around existing conditions in orderto get more appropriate dot plot.

FIG. 2 depicts exemplary FACS analysis of GFP expression in fibroblastcells indicative of neutralizing antibody response induced with gB/pp65CMV VLPs in rabbits. Rabbits (n=6/group) were immunized (IM) twice atweeks 0 and 8 and bled 2 weeks later. Sera were pooled and tested atindicated dilutions in comparison to Cytogam™ at similar dilutionsagainst GFP-expressing CMV virus (TB40) in HFF fibroblasts. 100,000cells were collected during flow cytometric analysis of infected (GFP⁺)cells.

FIG. 3 depicts exemplary FACS analysis of GFP expression in fibroblastcells indicative of neutralizing antibody response induced with bivalentgB+gH CMV VLPs in rabbits. Rabbits (n=6/group) were immunized (IM) twiceat weeks 0 and 8 and bled 2 weeks later. Sera were pooled and tested atindicated dilutions in comparison to Cytogam™ at similar dilutionsagainst GFP-expressing CMV virus (TB40) in HFF fibroblasts. 100,000cells were collected during flow cytometric analysis of infected (GFP⁺)cells.

Example 3: Exemplary Micro-Neutralization Assay for Detection ofNeutralizing Antibodies by Flow Cytometry

This Example describes detection of functional anti-CMV neutralizingantibodies by flow cytometry in sera samples from vaccinated animals.

Materials/Equipment

The following materials and equipment are used in this assay: HumanForeskin Fibroblasts (HFF-1) cells—ATCC#SCRC-1041; Human Arising RetinalPigment Epithelia—(ARPE-19)—ATCC#CRL-2302; Human herpesvirus 5 HCMV(UL32-EGFP-HCMV-TB40)—ATCC#VR-1578; Human CMV-GFP-Towne TS15-rR(obtained from Dr. M. McVoy, VCU-Virginia); Goat Antiserum to Rabbit IgG(GAR)—MP Cappel, Cat#: 55620; Complement sera from rabbit—Sigma-AldrichCat#S7764-5 ml; Standard Guinea pig complement—Cedarlane Cat#CL-5000;Sterile distilled water—Gibco, Cat#15230; Dulbecco's Modified EagleMedium (DMEM)—HyClone; Cat#SH30243.01 (growth media); Minimum EssentialMedium Eagle (MEM)—Sigma; Cat#M4655-500 ml (infectious media); Fetalbovine serum (FBS)—HyClone; Cat#SH30396.03; Penicillin StreptomycinSolution (Pen/Strep)—Sigma; Cat#P0781-100 ml; 1× Trypsin-EDTA—Sigma;Cat#T4174-100 ml; Modified phosphate buffered saline (DPBS—withoutCalcium and Magnesium)—HyClone; Cat#SH30028.02; Fixation buffer—BDBiosciences; BD Cytofix Cat#554655-100 ml; Cytogam(CMV-IGIV—Cytomegalovirus Immune Globulin Intravenous/human-CSL Behring;Commercial concentration 2.5 g/50 ml; Dimethyl sulfoxide(DMSO)—Sigma-Aldrich Cat#D1435-500 ml; Biosafety cabinet; Incubator 5%CO2, 37° C.; Centrifuge; Vortex; Sample acquisition tubes for a Flowcytometer (BD Falcon, 5 ml polystyrene round-bottom, REF 352054); 6-wellplates; Multichannel micropipette reservoirs; 5 and 10 ml graduatedpipettes; 10 μl to 1000 μl adjustable single channel micropipettes withdisposable tips; Cell scraper—NUNC; Cat#179707; 50 ml Falcon tubes—BD358206; Rotator—for eppendorf tubes; Fluorescence microscope; FACSCANmachine.

Obtaining and Harvesting the Virus

HFF-1 cells (95% confluent monolayer seeded in T150 flask) are infectedwith 1.5 ml of UL32-EGFP-HCMV-TB40 (“TB40”). ARPE-19 cells are infectedwith HCMV-GFP-Towne-TS15-rR (“Towne”) in the same manner. Since theseviruses are light sensitive, cells are infected with the light off. Theflasks are incubated in a CO2 incubator/37° C. for 30 minutes to allowattachment between cells and virus.

25 ml of infectious media is then added to the cells. (see Tables 6 and7). Flasks are kept in CO2 incubator/37° C. until cells are wellinfected (in a case of TB40, swelling and rounding cells are seen bylight microscope or green fluorescence with fluorescent microscope; forTowne, infection is detected by green fluorescence). Time necessary forgood infection depends on the infectious capacity of the ascendantvirus.

Harvesting is started by removing almost the entire supernatant from theflask and keeping it sterile in a 50 ml Falcon tube. Residualsupernatant, around 5 ml, facilitates scraping of the cells with a cellscraper. Good scraping and strong pipetting up and down (up to 10×)enables virus to release from the cells. 10 ml of the originalsupernatant volume from the Falcon tube is added and spun down at 900rpm for 10 minutes. The pellet is removed and around 15 ml ofconcentrated virus is kept. 5% of cryoprotectant (DMSO) in total amountof virus is added. 1 ml aliquots are prepared, labeled, and kept at −80°C. for short time or in liquid nitrogen for long period of time.

TABLE 6 HFF-1 cells growth and infection media for CMV-GFP-TB40Concentration Reagent Supplier Cat# of components Volume HFF-1 growthmedia DMEM HyClone SH30243.01 80% 420 ml (DMEM + 15% FBS + (Dulbecco'sModified 1% Pen/Strep) Eagle Medium) FBS (Fetal bovine HycloneSH30396.03 15%  75 ml serum) Pen/Strep (Penicillin Sigma P0781-100 ML 1% 5 ml Streptomycin Solution) HFF infectious MEM Sigma M4655-500 ml 94%470 ml media for CMV- (Minimum Essential GFP-TB40 virus Medium Eagle)(MEM + 5% FBS + FBS (Fetal bovine Hyclone SH30396.03 5%  25 ml 1%Pen/Strep) serum) Pen/Strep (Penicillin Sigma P0781-100 ML 1%  5 mlStreptomycin Solution)

TABLE 7 ARPE-19 cells growth & infection media for CMV-GFP-TowneConcentration Reagent Supplier Cat# of components Volume ARPE-19 growthDMEM:F-12 HyClone SH30023.01 80% 420 ml media (Dulbecco's Modified Eagle(DMEM:F-12 + Medium nutrient mixture F-12 15% FBS + HAM) 1% Pen/Strep)FBS (Fetal bovine serum) Hyclone SH30396.03 15%  75 ml Pen/Strep(Penicillin Streptomycin Sigma P0781-100 ML 1%  5 ml Solution) ARPE-19DMEM:F-12 HyClone SH30023.01 99% 470 ml infection media (Dulbecco'sModified Eagle for Towne Medium nutrient mixture F-12 growth HAM)(DMEM:F-12 + FBS (Fetal bovine serum) Hyclone SH30396.03 5%  25 ml 5%FBS + 1% Pen/Strep (Penicillin Streptomycin Sigma P0781-100 ML 1%  5 mlPen/Strep) Solution)

Experimental Set-Up and Method

Pre-bleed and Post-immunized serum samples are heat inactivated at 56°C. for 30 minutes prior to use. Commercial sera, Cytogam, that serves aspositive control is heat inactivated, as well. Serum dilutions are madestarting from ⅙ up to desired dilutions in duplicates. Cytogam is testedat comparable dilutions, a prior diluting the stock reagent 1:20 toadjust to the Ig content of human/rabbit sera (see Table 8).

TABLE 8 Sera and virus dilutions (as an example 1in2 virus dilution isshown) Media without FBS Working to make Sera volume Neat virus seradilutions Total in assay in assay Final concentration dilution Seravolume (μl) (μl) (μl) (μl) Sera vs. Virus ⅓ 100 μl neat sera 200 300 130μl ⅓ 130 ⅙ vs. ½ ⅙ 150 μl 1⅓ 150 300 130 μl ⅙ 130 1/12 vs. ½ 1/12 150 μl1⅙ 150 300 130 μl 1/12 130 1/24 vs. ½ 1/24 150 μl 1/12 150 300 130 μl1/24 130 1/48 vs. ½ 1/48 150 μl 1/24 150 300 130 μl 1/48 130 1/96 vs. ½1/96 150 μl 1/48 150 300 130 μl 1/96 130 1/192 vs. ½ 1/192 (etc.) 150 μl1/96 150 300 130 μl 1/192 130 1/384 vs. ½(etc.)

Particular concentrations of sera (Cytogam) and virus are combined androtated at 37′C for 1 hr. In some assays, complement is included. Forassays utilizing complement and HFF cells, 10% standard guinea pigcomplement is added to each particular sera (Cytogam)/virus mixture.Virus control is treated the same way. For assays utilizing complementand ARPE-19 cells, 2.5% rabbit complement is added to each mixregardless which species is used (rabbit or sera). The sera and virusare rotated at 37′C for 1 hr.

95% confluent cells (seeded in 6-well plates) are carefully rinsed twicewith warm PBS prior to virus-sera mixture application, in duplicate. 100μl of mixture/well+200 μl infection media is added to avoid drying over4 hours of incubation. Cells are incubated at 37° C./5% CO2/4 hrs (withrocking every 60 minutes). After a 4-hour incubation, contents from eachwell are carefully aspirated with a pipette or poured off, and 3 ml ofappropriate, fresh, warm infectious media is added. Plates are incubatedat 37° C./5% CO2 until well infected. 5 days after incubation, cells areanalyzed for virus infection. Infection of HFF-1 cells is determined byswelling and rounding of cells, visualized by light microscope or greenfluorescence with a fluorescent microscope. ARPE-19 cell infection isdetected as green fluorescence with a fluorescent microscope.

Sample Collection and Preparation for Flow Cytometry

Before sample collection, the presence of good infection is confirmedunder a light microscope and, if available, by analyzing GFP integratedinto cells using a fluorescent microscope. Media is aspirated or pouredoff from each well. Cells are rinsed twice with PBS (HyCloneDPBS/modified without Calcium and Magnesium; Cat#SH30028.02). To HFF-1cells are added 100 μl of 1× Trypsin-EDTA (Sigma; Cat#T4174-100 ml) and200 μl of PBS. To ARPE-19 cells are added 200 μl of 1× Trypsin-EDTA and100 μl of PBS. Cells are kept in a CO2 incubator 2-3 minutes until cellsare well trypsinized. 1 m/well of PBS+5% FBS are added to stop trypsin.Cells are collected into transparent flow intended tubes (two wells forsame sample/tube) (BD Falcon, 5 ml polystyrene round-bottom, REF352054). Wells are visualized under a light microscope and if cellsremain, an additional 500 μl PBS+5% FBS is added to collect additionalcells.

Falcon tubes are spun down at 900 rpm for 10 minutes. The supernatant isdiscarded and the pellet is kept. The tube is vortexed to disperse thepellet in the residual buffer. The remaining steps are performed in thedark. 200 ul Cytofix (BD Biosciences, BD Cytofix, Fixation bufferCat#554655 100 ml) are added to each tube and samples are incubated onice for 15 minutes. 1 ml of mixture PBS+5% FBS is added to each tube tostop Cytofix. Tubes are spun down at 900 rpm for 10 minutes. Thesupernatant is poured off and the pellet is kept. The pellet isreconstituted in 500 ul of PBS+5% FBS, vortexed vigorously, and flowcytometry is performed.

Example 4: Neutralization Activity of Exemplary VLPs in Rabbits

CHO cells were transfected at a cell density between 1.5E06 to 2.0E06cells/mL with plasmids of monovalent gB-G and monovalent gH-G (preparedas described in PCT/US2012/64556). Stuffer DNA was added to make totalDNA concentration up to 1 μg/mL cell culture. The plasmids used fortransfection were first purified by MaxiPrep or GigaPrep plasmidpurification kits (Qiagen). The PEIMAX used for transfection to deliverDNA to the cells was provided at a ratio of 6:1 (PEI: DNA wt/wt). Thecell culture was harvested 72 hours post transfection by centrifuging at4000 rpm for 20 minutes, using rotor JS-4.2A by Beckman Coulter, in 1Litre bottles. The supernatant was filtered through 0.8/0.45 μm filter(AcroPak 500 Capsule, Pall). The filtered supernatant was thenconcentrated by Tangential Flow Filtration (TFF for concentration ofVLPs) and diafiltered against histidine-containing buffer. 70 μL ofBenzonase (Novogen 99% purity (D00127703) containing 250 Units/μL) wassuspended in 400 μL of PBS that was supplemented with MgCl₂ to have a 2mM final concentration. TFF retentate portions were mixed with dilutedBenzonase in PBS solution and kept in a rotary shaker (head-to-toerotation) at room temperature for 1 hour and then the tubes were placedat 4° C. overnight. The diafiltered benzonase treated material was thenloaded onto an anion exchange chromatography column (AEX for reductionof DNA and host proteins) where the flowthrough was collected. Theflowthrough was then sterile filtered through 0.45 μm and aliquoted indifferent volumes.

Monovalent gB-G and monovalent gH-G VLP compositions prepared asdescribed were mixed together, adjuvanted with alum and then tested infemale New Zealand White rabbits 6-8 weeks old (minimum 5 animals pertest group) for neutralizing activity using the microneutralizationassay described in Example 3. Rabbits were immunized intramuscularlywith 0.5 ml (250 μl in two sites of the proximal caudal hind thighmuscle) of VLP compositions three times, once on day 0 (Prime) and onceon day 57 (week 8 Boost) and once on day 162 (week 24 Boost). Rabbitswere treated with 50 μg of both monovalent gB-G and monovalent gH-G(mixed together at a 1:1 ratio) VLP composition. To assess humoralimmune responses in rabbits, blood was collected from all rabbits in thestudy pre-1 st immunization and then post-1 st immunization days 28, 42and 55 and post-2nd immunization at day 14.

Neutralizing antibody responses to HCMV were determined using amicroneutralization assay in fibroblast cells based on a GFP-expressingCMV virus (TB40) and flow cytometric analysis of infected (GFP+) HFF-1cells as previously described. Rabbit sera collected pre- andpost-immunizations as described were pooled and tested for neutralizingactivity in the presence of guinea pig complement against HCMVexpressing GFP in HFF fibroblasts relative to a positive control CMVhyperglobulin, Cytogam™ and a negative control consisting of empty GagVLP (lacking antigenic proteins gB-G and/or gH-G).

FIG. 4 shows the percent neutralization in HFF-1 cells incubated withCMV-GFP-TB40-010512 virus in presence of 10% Guinea Pig complement andrabbit serum (group 7 pooled sera from 15RA09 was used as a positivedata representative where animals were treated with monovalent gB-G andmonovalent gH-G VLPs three times and also group 8 from 15RA05 study(empty Gag) was used as a negative control representative). As shown inFIG. 4, the combination of monovalent gB-G and monovalent gH-G VLPcomposition elicited a rapid, synergistic sustained neutralizingantibody response in rabbits against fibroblast cell infection, whilethe empty Gag VLP showed no neutralizing antibody response.(“GFP-TB40-010512” denotes Human herpesvirus 5 HCMV(UL32-EGFP-HCMV-TB40)—ATCC#VR-1578 (described in Example 3) grown on May1, 2012.)

Pooled rabbit sera was also tested for neutralizing antibody responsesto HCMV using a microneutralization assay in epithelial cells based on aGFP-expressing HCMV virus (Towne TS15-rR) and flow cytometric analysisof infected (GFP+) ARPE-19 cells as previously described. Rabbit seracollected pre- and post-immunizations as described were pooled andtested for neutralizing activity in the presence of 2.5% rabbitcomplement against HCMV expressing GFP in ARPE-19 epithelial cellsrelative to a positive control CMV hyperglobulin, Cytogam™ and anegative control consisting of empty Gag VLP (lacking antigenic proteinsgB-G and/or gH-G).

FIG. 5 shows the percent neutralization in ARPE-19 cells incubated withCMV-GFP-Towne-150612 virus in presence of 2.5% rabbit complement andrabbit serum (group 7 pooled sera from 15RA09 was used as a positivedata representative where animals were treated with monovalent gB-G andmonovalent gH-G VLP compositions three times and also group 8 from15RA05 study (empty Gag) was used as negative control representative).As shown in FIG. 5, the combination of monovalent gB-G and monovalentgH-G VLP composition elicited a rapid, synergistic sustainedneutralizing antibody response in rabbits against epithelial cellinfection while the empty Gag VLP showed no neutralizing antibodyresponse. (“GFP-Towne-150612” denotes Human CMV-GFP-Towne TS15-rR(obtained from Dr. M. McVoy, VCU-Virginia, and described in Example 3)grown on Jun. 15, 2012.)

Other Embodiments

Other embodiments of the disclosure will be apparent to those skilled inthe art from a consideration of the specification or practice of thedisclosure disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope of thedisclosure being indicated by the following claims. The contents of anyreference that is referred to herein are hereby incorporated byreference in their entirety.

1.-30. (canceled)
 31. A method comprising steps of: (i) mixingheat-inactivated serum from a subject that has been immunized with anHCMV candidate vaccine with an HCMV comprising a fluorescent moiety toform a mixture, wherein the HCMV comprising the fluorescent moietycomprises a gH/gL/gO complex; (ii) adding 10% guinea pig complement tothe mixture from (i); (iii) contacting a host cell that is susceptibleto infection by HCMV, wherein said host cell is a human foreskinfibroblast cell of cell line HFF-1, under conditions that allowinfection with the mixture of heat-inactivated serum in the presence ofrabbit complement of (ii); (iv) assessing a fluorescence level of thehost cell that has been contacted with the mixture by flow cytometry;and (v) determining a level of infection of the host cell based on theassessed fluorescence level.
 32. The method of claim 31, wherein thesubject that has been immunized is a human.
 33. The method of claim 31,wherein the serum comprises anti-HCMV neutralizing antibodies.
 34. Themethod of claim 31, wherein the HCMV candidate vaccine comprises VLPs.35. The method of claim 34, wherein the VLPs comprise one or more of gB,gH, and pp65 from HCMV.
 36. The method of claim 31, wherein the mixtureof (ii) is incubated for at least 15 minutes, at least 30 minutes, atleast 1 hour, or at least 2 hours before step (iii).
 37. The method ofclaim 31, wherein the assessing step (iv) is performed in a highthroughput manner.
 38. The method of claim 31, wherein the assessingstep (iv) comprises comparing the fluorescence level to a reference. 39.The method of claim 38, wherein the reference is a historical reference.40. The method of claim 38, wherein the reference is a side-by-sidereference.
 41. The method of claim 31, further comprising determining ananti-HCMV neutralizing antibody titer in the serum based on thedetermining step.
 42. The method of claim 41, further comprisingevaluating the efficacy of the candidate HCMV vaccine based on thedetermining step.
 43. The method of claim 42, further comprisingselecting the candidate HCMV vaccine as a vaccine that inducesneutralizing antibodies if the fluorescence level of the host cell thathas been contacted with the mixture is lower than a referencefluorescence level.
 44. The method of claim 31, wherein the contactingstep (iii) comprises an incubation of at least 1 hour, at least 2 hours,at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours,at least 7 hours, or at least 8 hours.
 45. The method of claim 31,wherein the host cell is further monitored for infection by visualcellular morphology assessment or visual fluorescence assessment. 46.The method of claim 45, wherein the visual cellular morphologyassessment comprises monitoring for cellular swelling and rounding. 47.The method of claim 45, wherein the fluorescence assessment is conductedusing a fluorescence microscope.
 48. The method of claim 31, wherein afluorescence level of the nucleus of the host cell is assessed.
 49. Themethod of claim 31, wherein a fluorescence level of the whole host cellis assessed.
 50. The method of claim 31, further comprising a step ofquantitating the fluorescence level of the host cell.